The present invention relates to the modification of cellulose normally synthesized by cellulose-producing microorganisms. This modification results from the selection of mutant microrganisms which form cellulose II rather than the usual native cellulose (cellulose I).
Cellulose is produced be a variety of microorganisms including the genera Acetobacter, Rhizobium, Alcaligenes, Agrobacterium, and Sarcina (see, for example, Deinema and Zevenhuizen, 1971 or Brown, et al., 1983). The growth of these cellulose-producing microorganisms with the production of cellulose occurs when said microorganisms are cultivated in an appropriate nutrient medium.
Appropriate nutrient media of the present invention generally include standard nutrient media such as GYC which contains (g/liter of distilled water): yeast extract, 10.0: D-glucose, 50.0; CaCO.sub.3, 30.0, and agar, 25. Various alternatives such as replacements for glucose or yeast extract, and the omission of agar or CaCO3 are usable and well known to those skilled in the art (De Ley et al., 1984). The preferred nutrient medium used directly or with modifications described herein was that first described by Hestrin and Schramm (Hestrin and Schramm, 1954). A standard Schramm-Hestrin medium (SH medium) used herein contained (g/L): D-glucose, 20; peptone, 5; yeast extract, 5; 5; dibasic sodium phosphate, 2.7, and citric acid monohydrate, 1.15. The pH of the medium may be adjusted to between 3.5 and 5.5 with HCl or it may be used without adjustment.
The cellulose produced by Acetobacter xylinum (formerly known as Acetobacter aceti subsp. xylinum and reclassified in the 1984 Bergey's Manual (DeLey, et al., studied. In the present application, the primarily studied cellulose-producing microorganism is termed "Acetobacter xylinum" (A. xylinum). It is understood that these several names may be used to indicate the same organism.
Alterations of the cellulose fibrils produced by microorganisms by agents such as carboxymethyl cellulose (CMC) or by dyes such as Congo Red or Tinopal have been previously observed by electron microscopy (see, for example, Haigler and Benziman, 1982). However, nowhere before the present invention has any substance or method been found or suggested to initiate the microbial production of cellulose II. (Chemical conversion of cellulose I to Cellulose II after synthesis is, however, widely known.)
Acetobacter xylinum (now classified under A. pasteurianus and A. hansenii in De Ley et al., 1984) is a gram negative aerobic bacterium that produces an extracellular ribbon of cellulose (Brown et al., 1976). When grown in stationary liquid culture, the ribbons of cellulose produced by individual cells entangle, eventually forming a thick mat of cellulose called a pellicle at the surface of the medium. Although all cellulose is a beta-1,4 linked polymer of D-glucopyranose, it occurs in several distinct crystalline forms known as allomorphs or polymorphs (briefly reviewed in Sarko, 1978). Five different cellulose allomorphs are currently known: cellulose I, cellulose 11, cellulose 111, cellulose IV, cellulose X. Nearly all organisms which synthesize cellulose, including A. xylinum, produce the allomorph cellulose I or "native cellulose". Although a few organisms have been claimed to synthesize cellulose II, the present invention involves the first confirmed description of in vivo synthesis of cellulose II by any prokaryote, particularly Acetobacter.
Variability within the Acetobacter genus has been extensively discussed in the microbiological literature. Some workers (e.g., Shimwell and Carr, 1960) have reported that both biochemical and colony morphology mutations were "extraordinarily facile" and of "kaleidoscopic rapidity and effect". In contrast, Schell and De Ley (1962) state that the rate of mutation is comparable to other genera of bacteria. These conflicting beliefs are reflected in two different concepts about what constitutes the Acetobacter genus. On one hand, workers such as Shimwell have proposed that all acetic acid bacteria are merely strain variations within two biotypes Gluconobacter oxydans and Acetobacter aceti. On the other hand De Ley (1961) and others have proposed that a phylogenetic derivation of species whereby all extant strains have been derived from ancestral strains primarily by the deletion of various enzymes.
It has been reported that many strains of Acetobacter can lose the ability to produce cellulose. (Reversion of putative "non-cellulose" producers has also been claimed, e.g. Shimwell, 1956). Such cel- variants can arise spontaneously, or be induced by various physical or chemical treatments.
Cellulose deficient Acetobacter strains (referred to in this paper as "cel-" strains) were reported in 1954 by Schramm and Hestrin. These workers noted that when serially transferred in shaken liquid cultures, A. xylinum loses its ability to synthesize cellulose. Such changes were generally correlated with changes in colony morphology of cells grown on nutrient agar plates (i.e., the formation of smooth colonies). Schramm and Hestrin proposed that shaking cultures conferred a selective advantage to naturally occurring cellulose deficient mutants within a population leading to an eventual loss of the wild type. The wild type, however, was selected for in static liquid cultures because the cellulose pellicle formed at the surface of the medium, kept wild type cells near their oxygen source.
Following this first report, cellulose deficient forms have been isolated from many strains by numerous methods. Creedy et al. (1954) isolated cellulose deficient mutants of A. acetigenum by treatment with sodium arsenate, while Steele and Walker (1957) found that variations in culture medium (notably the use of ethanol as a carbon source) was also correlated with the loss of cellulose production. Since this time, other reports of cel- forms have appeared. It is not clear if these agents act as mutagens or if they act merely as selection media for spontaneously occurring variants.
Some evidence suggests that "cellulose deficient" mutants of Acetobacter may actually produce small amounts of cellulose. Valla and Kjosbakken (1982) reported the presence of "cel-" mutants which have alterations in the gene(s) involved in cellulose synthesis, In the same paper, these authors report that cel- cells can form aggregates that are dispersed by cellulase. In addition, they report that 50% of these mutants can be induced to form cellulose by treatment with tetracycline. (The allomorph that these mutants produce was not determined.) It is suggested that the mutation is not in the structural gene(s) for cellulose biosynthesis, but in some other accessory gene necessary for complete expression of the cel+ phenotype.
In 1977, Forge published a study on "non-pellicle forming" strains of A. xylinum isolated from shake cultures. While these cells do not form a pellicle, Forge claimed to have identified short fibrils associated with the outer membrane using freeze-etch electron microscopy. Although elongated structures are present in his micrographs, these appear to be very similar to plastic deformation artefact (Sleytr and Robards, 1977). Forge also examined non-pellicle forming cultures with x-ray diffraction. In a few cases, a faint cellulose I pattern was observed. Forge concluded that while his strain did not form a pellicle, small amounts of cellulose I were synthesized, perhaps by enzymes that had been dispersed over the entire membrane surface. This is the only report known to the Applicants that discusses the x-ray diffraction pattern produced from non-pellicle forming cultures.
As mentioned, the cellulose molecule is defined not only by its chemical constitution, beta-1,4 linked glucopyranose, but also by the manner in which these molecules are packed together into crystalline forms. Currently, five distinct crystalline allomorphs of cellulose have been identified by their x-ray diffraction patterns: cellulose I,II,III,IV, and X (e.g., Ellefsen and Tonnesen, 1971). These allomorphs may also be grouped according to chain polarity, as described later. The unique packing of glucan chains into cellulose crystallites can be identified by a variety of techniques. Older cytochemical methods for distinguishing cellulose allomorphs using iodine staining (e.g., Roelofsen, 1959) are notoriously unreliable and have been replaced by other methods which yield unique "fingerprints" for cellulose I and II and thus allow these allomorphs to be distinguished. The distinctive infrared (IR) spectra of cellulose I and II are described in Blackwell and Marchessault (1971). As described by these authors, "For cellulose II . . . the -OH stretching region is distinctively different from the equivalent region in cellulose I. This allows for easy identification of the two forms" (p.17). Crosspolarized magic angle spinning 13C nuclear magnetic resonances (CP/MAS 13C NMR) provides another method for the identification of cellulose allomorphs. As described in Horii et al. (1987), ". . . the C1 and C4 resonances [of regenerated cellulose] split into doublets with equivalent intensities. This is in contrast to the case of native cellulose, reflecting that the crystal structure of regenerated cellulose is cellulose II" (p. 128). Raman spectroscopy may also be used to distinguish the allomorphs of cellulose. For example, Wiley and Atalla (1987) note that although the spectra of cellulose I samples derived from various sources may vary somewhat,". . . in the spectrum of cellulose II . . . the frequency and number of peaks is significantly different" (p. 164 and spectra p. 162). Electron diffraction has been used to study crystalline materials in general, and the various cellulose allomorphis in particular (French, 1985). The technique of neutron diffraction is theoretically also suited for cellulose crystal studies (French, 1985). The most widely used technique is x-ray diffraction; it allows the determination of distances separating planes of atoms (called d-spacings) in the crystals. These d-spacings are unique for any given crystal form and thus act like a fingerprint for each of the cellulose allomorphs (Tripp and Conrad, 1972). Actual d-spacing values vary somewhat from sample to sample, especially for larger values. Short, general reviews of cellulose polymorphism can be found in Tripp and Conrad (1972), Sarko (1978) and Ellefsen and Tonnesen (1971).
Of the cellulose allomorphs, cellulose I ("native cellulose") is the form found naturally occurring in nearly all plants. Cellulose II, also known as "hydrate" cellulose is well known industrially. For example, both rayon and mercerized cellulose are forms of cellulose II. It can be generated by precipitation from solution, by treatment of cellulose I with strong swelling agents such as 24% KOH, and other means. Cellulose I and cellulose II are the commercially dominant allomorphs of cellulose.
The glucan chains which comprise native cellulose (cellulose I) are thought by most workers to exist in a "parallel" orientation (FIG. 1), i.e. all the reducing ends of a glucan chain represented by arrowheads in the diagram are pointed in the same direction. In contrast, most workers believe that cellulose II exists in an antiparallel form (reducing ends of the glucan chains having a statistically random orientation within a fibril). This prevailing idea is not universally accepted. French (1985) has pointed out that the R values (measures of fit of a given model with x-ray diffraction data) for a parallel and antiparallel chain cellulose I do not completely eliminate either model of chain polarity. Hieta, et al. (1984) have recently shown by cytochemical means that all the reducing ends of Valonia microfibrils are clustered on one end of the fibril, thus lending support to the idea of a parallel chain arrangement for cellulose I in this organism. Sakthivel, et al. (1987) have recently proposed that both a parallel and antiparallel cellulose II may exist.
Several allomorphs of cellulose exist in two subforms (e.g. III.sub.I and III.sub.II) depending on which "parent" allomorph gave rise to it (Sarko, 1978). Thus, the parallel form of cellulose III (generated from cellulose I) is designated III.sub.I, while the antiparallel form (made from cellulose II) is designated III.sub.II. Although the defraction patterns of these subforms are indistinguishable, they can be identified because if the transition is reversed, only the original parent will be produced. This property is referred to as "memory".
Interconversion of the various cellulose allomorphs is summarized in FIG. 2. This figure also illustrates the irreversible transition of cellulose I to cellulose II. Cellulose II is the more thermodynamically stable form of cellulose, in part due to increased hydrogen bonding between stacks of glucan chains (French 1985).
Relative thermodynamic stability and chain polarity have important implications for the in vivo biosynthesis of cellulose. It is generally accepted that a single cellulose synthase enzyme will polymerize glucan chains unidirectionally, i.e., that all reducing ends will emerge from the enzyme pointing in the same direction. When numerous cellulose synthase enzymes are grouped together, such an arrangement has been suggested to facilitate the "cell directed self assembly" of a parallel chain polymer (Haigler and Benziman, 1982). This idea is implicit even in the earliest x-ray diffraction studies of cellulose structure; for example, Meyer and Mark (1928) originally proposed a parallel chain arrangement simply because they felt it was unlikely that two enzymes (one type synthesizing reducing end first, the other, nonreducing end first) could exist. It is of interest that the cellulose produced by essentially all plants (cellulose I) should be a thermodynamically less stable form of this polymer.
Over the last 50 years, a relatively small number of organisms has been suggested to make native cellulose II. These organisms ranged from bacteria to slime molds to algae. Each of these claims will be reviewed in the following section and are summarized in Table 1.
TABLE 1 ______________________________________ ORGANISMS SUGGESTED TO PRODUCE NATIVE CELLULOSE II Organism Citation ______________________________________ Halicystis grandis Sisson (1938, 1941) H. ovalis Sisson (1938, 1941) H. parlvula Sisson (1938, 1941) H. osterhoutii Sisson (1938, 1941) Oomyces sp. Frey-Wyssling (1976) Dictyostelium discoidium Raper & Fennell (1952) D. discoidium Gezelius and Ranby (1957) D. discoidium Muhlethaler (1956) Various green algae Nicolai & Preston (1952) Sarcina ventriculi Kreger (in Roelofsen, 1959) Enteromorpha intestinalis Dodson and Aronson (1978) ______________________________________
In 1938 and 1941, Sisson published studies on the cell walls of the green alga Halicystis. He suggested that the four species he examined, H. grandis, H. osterhoutii, H. ovalis, and H. parvula, contained the cellulose II allomorph although other crystalline material with a reflection around 13 Angstroms (A) (later ascribed to a crystalline 1,3-xylan; see Preston, 1974) was also present. These components were revealed only after sequential extraction with HCl followed by other solvents such as NaOH.
Halicystis has been subsequently investigated by several groups. Roelofsen et al. (1953), examined the walls of these cells and concluded that a xyloglucan (and not simply cellulose) was present. Frei and Preston (1961) Were able to isolate a cellulosic component from the wall, but felt that this material might result from chemical treatment causing crystallization of a previously "paracrystalline" component from the wall. Most recently, Huizing et al. (1979) reported that the walls of the gametophyte of Derbesia tennuissima (taxonomically equivalent to Halicystis) "failed to show any crystallinity by x-ray analysis, even after prolonged boiling in dilute acid". As summarized by Preston (1974), "it is still a moot point whether the glucan [from Halicystis] is cellulose II . . .".
In the 1950's naturally occurring cellulose II was reported to occur in a number of organisms. Raper and Fennel (1952) reported that intact sorophores of the cellular slime mold Dictyostelium discoidium gave weak fiber diagrams with two equatorial reflections at 4.05 A and 4.41A. Dictyostelium cellulose was also examined by Muhlethaler in 1956. Although untreated sorophores gave weak patterns, equatorial reflections of 4.03A, 3.33 A, and 2.56 A could be identified. Boiling the stalks in 5% H.sub.2 SO.sub.4 followed by 5% NaOH, however, resulted in the loss of these reflections and the appearance of reflections that appeared to correspond to cellulose I alone. Muhlethaler (I956) concluded that the high temperature cleaning treatment caused a conversion of cellulose II to cellulose I. (Contemporary ideas of cellulose thermodynamic stability make such a transition seem unlikely). Gezelius and Ranby (1957) examined two strains of this same genus. In stalks cleaned by boiling in 1N NaOH or by 1 N NaOH followed by boiling in 2.5 % H.sub.2 SO.sub.4, the major reflections of cellulose I together with a 7.17 A reflection attributed to cellulose II was observed. From this data, the authors concluded that cellulose was present as "a partly mercerized cellulose of very low crystalline order" and noted that "the problem will be further studied". In 1959, Gezelius published his observations on Acytostelium, an acellular slime mold. In untreated stalks from this organism, reflections of 10 A, 4.6 A and 3.85 A were observed. These reflections could not be attributed to any known cellulose allomorph. After purification by boiling IN NaOH, the diffraction pattern of native cellulose was obtained along with a persistent diffuse ring at approximately 10 A. Despite the fact that no reflections of mercerized cellulose were seen, the author stated that "the results agree with those obtained with Dictyostelium discoidium . . ." More recently, Blanton and Chanzy (1985) used electron diffraction to demonstrate that the stalk of Protostelium irregularis is composed of cellulose I.
During the 1950's, cellulose and other wall components of algae were also vigorously investigated. Nicolai and preston (1952) investigated nearly 60 species of green algae using a variety of techniques, and especially x-ray diffraction. Their results suggested that the algae investigated could be placed into three groups. The cell walls of the first group were composed of highly crystalline cellulose I microfibrils usually arranges in the wall as cross parallel lamellae. Algae of this first group were found primarily in the order Cladophorales with a few representatives from the order Siphonales.
The second group contained the bulk of the algae examined. The walls of this group contained poorly crystalline material whose d-spacings approximated those of cellulose II. The authors noted, however, that they could not rule out the possibility that this material was cellulose derivative and not cellulose II. (The hesitancy to conclude that cellulose II was absolutely present is emphasized by Cronshaw et al. (1958) when group 2 is defined as those algae certainly not containing cellulose I). The third wall group originally described by Nicolai and Preston was a small group of algae whose x-ray patterns were uninterpretable. At the time that these studies were carried out, the results were considered to follow taxonomic lines.
The presence of native cellulose II was also suggested for the red alga Griffithsia flosculosa (Myers et al., 1955) although this contention was subsequently moderated (Myers and Preston, 1959).
The degree of caution expressed by Preston and coworkers on the occurrence of native cellulose II among the algae proved to be well founded. In 1963, Frei and Preston showed that some of the putative "cellulose II" reflections observed in previous studies persisted even after ashing. (Ashing would destroy any of the cellulose allomorphs). It was then observed that small clay particles adhering to the walls of algae collected from nature exhibited x-ray reflections similar to those of cellulose II. In addition, the flat clay platelets lay orientated on the cell walls, a fact which also explains why the x-ray patterns reported in previous papers were arranged in uniplanar orientations. (Such clays exist as small platelets and thus would not be present for material cultured in natural seawater that had been filter sterilized through 0.22 micron filters).
Although Frei and Preston (1963) did not reexamine all the species discussed in previous reports, the identification of clays on algal walls renders the identification of cellulose II by Nicolai and Preston (1952), and all others using walls collected from nature, uncertain. This concern extends even to material which has been closely examined for contaminants or has been soaked overnight in dilute acids to remove mineral incrustations. However, it is not possible to completely dismiss all of these claims since it was reported that the alga Enteromorpha intestinalis, cultured in the absence of such clay particles, may contain cellulose II (Dodson and Aronson, 1978).
Synthesis of cellulose II by a Prokaryote has also been suggested. Kreger, cited in Roelofsen (1959), examined "mechanically cleaned walls" (thus, apparently avoiding the problems of chemically induced conversion of cellulose I to cellulose II) from the bacterium Sarcina ventriculi. This material was claimed to show a cellulose II x-ray diagram, although this data was apparently never published. Chemical and enzyme digestion studies by Canale-Parola et al. (1961) and Canale-Parola and Wolfe (1964) strongly suggest that cellulose is associated with the bacterial packets, although the allomorph was not determined. Subsequent studies in the laboratory of the Applicants suggests that S. ventriculi strains obtained from the American Type Culture Collection of bacteria (ATCC) do produce the cellulose II allomorph.
Finally, it should be noted that in vitro cellulose synthesis by cell-free preparations of Acetobacter xylinum have recently been shown to produce the cellulose II allomorph (Bureau and Brown, 1987). This presumably results from the disruption of closely associated cellulose synthase enzymes and the consequent loss of cell directed assembly of the crystalline cellulose ribbon. In the absence of such control, the thermodynamically stable allomorph will form. Until this time, however, it has never been suggested that intact cells of Acetobacter can produce cellulose II. Commercial Production of Cellulose II
The primary allomorphs of cellulose used industrially are cellulose I and cellulose II. Commercial products composed of cellulose II or substantial fractions thereof) include "rayon" and "mercerized cellulose". Both rayon and mercerized cellulose are manmade products derived from natural cellulose I such as that from wood pulp or cotton.
Some of the major processes for manufacturing cellulose II include the viscose process, the cuprammonium process, and the saponified cellulose acetate process (see Joseph, 1977; Moncrieff, 1975, or related textbook). These are briefly described below.
The viscose process involves steeping cellulose I (e.g., wood pulp) in alkali solution to form soda cellulose. This material is shredded, aged, and then treated with carbon disulfide which forms a bright orange intermediate called sodium cellulose xanthate. The xanthate is then dissolved in dilute NaOH, aged, and extruded through a spinerette system into an acid bath to form fibers of cellulose II. (See Moncrieff, 1975).
In the production of cuprammonium rayon, cellulose is bleached and dissolved in a solution of ammonia, copper sulfate and NaOH. This viscous blue solution is then spun into water, acidified and cleaned. The resulting cellulosic fibers consist of cellulose II.
Another method that has been used to produce highly crystalline cellulose II involves the deacetylation of cellulose acetate. This saponified cellulose process begins with cellulose acetate treated with alkali. The acetyl groups react to form sodium acetates which then split off. They are replaced by hydroxyl groups leaving cellulose II.
Mercerization is a chemical finishing process for cellulose textiles in which cellulose I is converted into cellulose II. This process, consists of treating wetted fabric with alkali (typically 16-27% NaOH) and, after a suitable amount of time, washing the fabric.
Each of the processes described above ultimately involve the conversion of cellulose I to cellulose II. In general, cellulose II products have improved dye uptake, higher strength, and greater moisture absorbance than the cellulose II of cotton fibers (Joseph, 1977). The actual I5 properties of cellulose II fibers are dependent on how they are produced and processed. Advantages of Native Synthesis of Cellulose II
The present invention involves the native synthesis of cellulose II by Acetobacter xylinum. Although the organism described in the current report does not produce large quantities of cellulose compared to the wild type, it does demonstrate that the native production of this allomorph in vivo can be accomplished. The production and identification of strains with high rates of sustained cellulose II production (and therefore industrial utility) has been established as a reasonable possibility.
The desirable properties of cellulose II derived by chemical treatment from typical cotton fiber cellulose I have already been mentioned. There are other advantages of in vivo production of cellulose II by A. xylinum (or similar organisms) as compared to current chemical production methods. As produced by Acetobacter mutants of the present invention, no chemical treatment is needed to convert cellulose I to cellulose II. In addition, unlike the wild type which may lose its ability to form cellulose upon prolonged agitated culture (including many conventional fermenters), the mutants described here appear to retain the ability to make cellulose II under these conditions. In addition, because the product does not consist of extended ribbons (i.e., there is no pellicle), it would be relatively easily to transfer the product to and from fermentation, cleaning and processing vessels. Finally, it seems reasonable to expect that mixed cultures producing both cellulose I and cellulose II (or even other allomorphs) could be produced.